Anticoccidial activity of metal chelates of substitutes bis-thiosemicarbazones of cyclic 1,2-diketones

ABSTRACT

Substituted bis-thiosemicarbazones of cyclic 1,2-diketones and metal chelates thereof effective in the control and treatment of coccidiosis.

United States Patent [72] Inventor Kenneth Butler Old Lyme, Conn. [21] Appl. No. 822,301 [22] Filed May 6, 1969 [45] Patented Nov. 23, 1971 [73] Assignee Pfizer Inc.

New York, N.Y. Original application Aug. 30, 1967, Ser. No. 671,527, which is a division of application Ser. No. 407,212, Oct. 28, 1964, now Patent No. 3,382,266, dated May 7, 1968. Divided and this application May 6, 1969, Ser. No. 822,301

[54] ANTICOCCIDIAL ACTIVITY OF METAL CHELATES OF SUBSTITUTES BIS- THIOSEMICARBAZONES OF CYCLIC 1,2- DIKETONES 4 Claims, No Drawings [52] U.S.Cl 424/289, 424/294, 424/295 [51] Int. Cl A6lk 27/00 [50] Field of Search 424/289, 294, 295

[56] References Cited UNITED STATES PATENTS 3,259,643 7/1966 Nash 424/287 Primary Examiner-Sam Rosen Attorney-Connolly and Hutz ABSTRACT: Substituted bis-thiosemicarbazones of cyclic 1,2-diketones and metal chelates thereof effective in the control and treatment of coccidiosis.

ANTICOCCIDIAL ACTIVITY OF METAL CHELATES OF SUBSTITUTES BIS-THIOSEMICARBAZONES OF CYCLIC l ,2-DIKETONES CROSS-REFERENCE TO RELATED APPLICATIONS This application is a division of application Ser. No. 671,527 filed Aug. 30, 1967, now U.S. Pat. No. 3,497,600 of Feb. 24, 1970, which in turn is a division of application Ser. No. 407,212 filed Oct. 28, 1964, and now US. Pat. 3,382,266 ofMay 7, 1968.

This invention relates generally to new chemical compounds. It relates further to new compounds which are useful in treating and preventing the poultry disease coccidiosis. More particularly, it is concerned with novel substituted bisthiosemicarbazones of cyclic l,2-diketones and nuetral metal chelates thereof and their efiectiveness in the control and treatment of coccidiosis.

Coccidiosis, a poultry disease, is caused by several species of protozoan parasites of the genus Eimeria, such as E. tenella, E. necatrix, E. acervulina, E. maxima, E. hagani and E. tenella. E. renella is the causative agent of a severe and often fatal infection of the ceca of chickens which is manifested by exten sive hemorrhage, accumulation of blood in the ceca, and the passage of blood in the droppings. Essentially, coccidiosis is an intestinal disease which is disseminated by birds picking up the infectious organism in droppings on contaminated litter or ground. By damaging the intestinal wall, the host animal is unable to utilize its food, goes off its feed and in untreated cases the disease terminates in either the death of the animal or the survival of unthrifty birds known commonly as culls.

It has now been discovered that certain substituted bisthiosemicarbazones of cyclic 1,2-diketones and metal chelates thereof possess a high degree of activity against protozoa which cause coccidiosis. It is one object of the present invention to provide such compounds. A further object is the provision of a method which is useful in the treatment or prevention of coccidiosis which comprises the utilization of the herein disclosed novel compounds. Further objects will become evident from the ensuing detailed description of the invention.

The novel substituted bis-thiosemicarbqzones of cyclic 1,2- diketones disclosed herein may be represented by the following structural formula:

wherein R is selected from the group consisting of H, alkyl containing from one to carbon atoms, chloro, hydroxy, mercapto and amino; R represents a member of the group consisting of H, and alkyl containing from one to eight carbon atoms; n is an integer from two to [8; and R is hydrogen or alkyl from one to eight carbon atoms. The corresponding neutral metal chelates of the above-described compounds may be indicated by the following formula:

wherein M is a divalent cation selected from the group consisting of Fe, Zn Sn, Co, Cu and Ni.

In order to prepare the chelates encompassed by formula II from the corresponding bis-hydrazone compound, it is apparent that substituent R in formula I of the corresponding hydrazone compound must be hydrogen. Said requirement is based on the necessity of forming the enol form of the thio ketone in order that chelation may take place.

The procedure for preparing the novel substituted bisthiosemicarbazones of cyclic 1,2-diketones of this invention involves the condensation of a cyclic 1,2-diketone or a substituted derivative thereof with two equivalents of a thiosemicarbazide derivative under acidic conditions. By the term thiosemicarbazide derivative" is meant a compound which is represented by the following formula:

wherein R is a member selected from the group consisting of H and alkyl containing from one to eight carbon atoms; and R is hydrogen or alkyl containing from one to eight carbon atoms. Suitable examples include thiosemicarbazide, N-4- methylthiosemicarbazide, N-Z-methythiosemicarbazide, etc. Such thiosemicarbazide derivatives are either well-known compounds or may be prepared using standard organic procedures familiar to those skilled in the art.

The reaction sequence is generally carried out in the following manner. The thiosemicarbazide derivative is dissolved in water made acid by the addition of HCl and added to a solution containing the cyclic-1,2-dione. The 1,2-diketone may be dissolved in a wide variety of solvents. For example, suitable organic solvents include methanol, ethanol, ether, acetic acid and the like. It is also possible to use an aqueous system or a mixture of water and one of the aforementioned organic solvents. The product which precipitates from said reaction mixture is filtered, dried and may be recrystallized from a suitable inert medium.

Since the bis-thiosemicarbazone, compounds are sought, it is required that at least two equivalents of thiosemicarbazide derivative reagent per equivalent of l,2-diketone be used. In fact, a slight excess of two equivalents is generally more preferred. The isolation step is a facile one in view of the fact that the product precipitates from the reaction mixture and can be easily removed by filtration.

The preparation of the neutral metal chelates of the substituted bis-thiosemicarbazones of cyclic l,2-diketones described above consists of a facile one step reaction procedure. An aqueous solution of the appropriate metal salt, for example, FeSO ZnCl SnCl CaCl,, CuCl or MCI; is added directly to a solution containing an equimolar amount of the cyclic bis-thiosemicarbazone compound. The workup involves filtering the precipitate which forms and drying said product by any number of suitable ways. In preparing the cyclic bis-thiosemicarbazone compound solution, a water-miscible solvent is utilized. For instance, dimethylformamide, dimethylsulfoxide, methanol, water and mixtures thereof are representative examples.

The cyclic 1,2-diketones utilized as starting materials in the herein disclosed invention are either well-known available reagents or their preparation well documented in the chemical literature. In addition, the preparation of substituted 1,2- diketones entails well-established procedures. For example, in preparing a-alkyl substituted cyclohexa-l,2-dione derivatives, cyclohexanone is converted to a 2-alkyl substituted cyclohexanone via an enamine derivative, and subsequently to the desired product by a selenium dioxide oxidation.

Further, in the cycloheptane series, successful 2-alkylation may be accomplished by alkylating the B-ketoester derivative prepared by the reaction of cycloheptanone with diethyl oxalate followed by a decarboxylation step and subsequent oxidation with selenium dioxide. Still further, 4-alkyl derivatives of l,2-dione may be obtained by reducing the corresponding 4- EXAMPLE 1 C :1; pentanel ,2-dione-bis-thiosemicarbazone To an ether solution containing 1,2-cyclopentanedione (7.0 g., 0.07 M) is added thiosemicarbazide (13.2 g., 0.15 M) dissolved in an aqueous HCl solution. The product which precipitates in a crystalline form, if filtered and recrystallized from a dimethyIformamide-H O system. The yield obtained is 14 g., 80.5 percent.

Analysis:

Calcd. for: C, 34.43; H, 4.95; N, 34.42; S, 26.21.

Found: C, 34.70; H, 4.95. N, 33.69; S, 26.16.

EXAMPLE ll Cyclohexanel ,2-dione-bis-thiosemicarbazone To an aqueous solution containing cyclohexane -l,2-dione (10.0 g., 0.09 M) is added thiosemicarbazide (23 g., 0.4 M) dissolved in an aqueous l-lCl solution. The crystalline product which precipitates is filtered and dried to give a yield of 10.5 g., 45 percent, M.P. 206-7C. dec.

Analysis:

Calcd. for: C, 37.21; H, 5.46; N, 32.55; S, 24.78.

Found: C, 38.03; 1-1, 5.47; N, 31.03; S, 24.15.

EXAMPLE 111 Cycloheptanel ,2-dione-bis-thiosemicarbazone To a solution containing cycloheptane-1,2-dione (15 g., 0.12 M) dissolved in ethanol (25 ml.) is added thiosemicarbazide (23.8 g., 0.26 M) dissolved in an aqueous HCl solution. The resulting reaction mixture is cooled, and the crystalline precipitate is filtered and dried. The yield of product is 23 g.,

80 percent, M.P. 189-190C. dec.

EXAMPLE 1V Cyclooctane l ,2-dione-bis-thiosemicarbazone To a solution containing cyclooctane-l,2-dione (6 g., 0.04 M) dissolved in acetic acid ml.) is added thiosemicarbazide (3.9 g., 0.04 M) dissolved in an aqueous HCl solution. The resulting solid is recrystallized from a dimethylformamide-water mixture to a crystalline product, 2.3 g., 19 percent, M.P. 177C. dec.

EXAMPLE V The bis-thiosemicarbazones of the following 1,2-diketones are prepared employing the procedure of example 1V wherein stiochiometric equivalent amounts of the corresponding 1,2- diketones are used in lieu of cyclooctane-lieu of cyclooctane- 1,2-dione and substantially the same results are obtained.

Thiosemicarbazlde derivative 1,2-diketone 3-ch1orocyc1ooctane-1,2-dione. 4-methy1cycl0hexane-L2-dlone 3aminocyclop entane-l,2-d1one Product Product cyclobutancl ,Z-dione-bisthioscmicurbuzone cyclododccanel .Z-dionc-bisthiusemicarbazone cycluhexadodecane- 1 .2-dionehis thioscmicarbuzone cyclooctadodecanel .2-dionehis-thiosemicarbazone 1.2-dikctune cyclobutanc-LZ-dionc cyclododecane-1.2-diune cyclohexadodccanel .Z-dionc cyclooctadudecunc-1,2-dione EXAMPLE V1 Cycloheptanel ,2-dione-bis-N-4-methylthiosemicarbazone 1 To a solution containing cycloheptane-l,2-dione (15 g., 0.12 M) dissolved in ethanol (25 ml.) is added N- .4methylthiosemicarbazide (21 g., 0.2 M) dissolved in an aqueous HCl solution. The resulting reaction mixture is filtered an the solid so obtained is crystallized from a methanol slurry. The product yield is 21 g., percent, M.P. 184 C. dec.

EXAMPLE V11 Cyclooctanel ,2-dione-bis-N-4-methylthiosemicarbazone To a solution containing cyclooctanel .2-dione 1.4 g., 0.01 M) dissolved in ethanol (30 ml.) is added N-4- methylthiosemicarbazide (2.1 g., 0.02 M) dissolved in an aqueous l-lCl solution. The crystalline precipitate which forms is filtered and dried,

The bis-N-4-methylthiosemicarbazones of the 1.2-diketones cited in example V are prepared by the above said procedure wherein stiochiometric equivalent amounts of said diketones are used in lieu of cyclooctanel ,2-dione and substantially the same results are obtained.

EXAMPLE V111 3-Methylcycloheptane-l ,Z-dione-bis-thiosemicarbazone To a methanolic (25 ml.) solution containing 3-methylcycloheptane-l,2-dione (8.0 g., 0.056 M) is added,

vthiosemicarbazide (10.3 g., 0.13 M) dissolved in an aqueous HCl solution. The crystalline precipitate which forms is filtered and dried to give 5.8 g., 36 percent of product, M.P. 202.SC. dec.

EXAMPLE 1X 4-Ethylcyclohexanel ,2-dione-bis-N-4-butylthiosemicarbazone To a methanolic solution (25 ml.) containing 4-ethylcyclohexane-l,2-dione (5 g., 0.036 M) is added N-4-butylthiosemicarbazide (11.7 g., 0.079 M) dissolved in an aqueous HCl solution. The solid which precipitates is filtered, dried' and crystallized from methanol to give 2.4 g., 22 percent of product, M.P. 103-108C.

EXAMPLE X Repeating the procedure of example IX, the following bisthiosemicarbazone derivatives are prepared using stoichiometric equivalent amounts of appropriate starting materials in lieu of Z-ethylcyclohexane-l ,2-dione and N-4-butylthiosemicarbazide and substantially the same results are obtained.

3-octyleyclopentane-1,2-dione-bis-N-2-methyl-tliiosemicarbazone.

4-dodecy1cyc10hexane-1,Z-dlone-l')is-N-2-0ctylth1osemicarbazone. 3-11ydroxycyclohexane-l,2-dione-bis-thiosomicarbazone. 3-mercaptocyc1ononane-l,2-dlone-bis-thiosernicarbazone.

EXAMPLE X1 EXAMPLE X]! L Cupric chelate of cycloheptanel,Z-dione-bis-thiosemicarbazone To a dimethylformamide solution (25 ml.) containing cycloheptane-l ,2-dione-bis-thiosemicarbazone (2.7 g., 0.01 M) is added an aqueous solution of CuCl 2H O (1.7 g., 0.01 M). The solid which precipitates is filtered and dried to give a 61 percent yield of product.

EXAMPLE Xlll Zinc chelate of cycloheptane-l,2-dione-bis-thiosemicarbazone To a dimethylformamide solution (40 ml.) containing cycloheptane-1,2-dione-bis-thiosemicarbazone (2.7 g., 0.01 M) is added an aqueous solution of ZnCl (1.4 g., 0.01 M). The solid which precipitates is filtered and dried to give a substantial yield of product.

EXAMPLE XlV EXAMPLE XV cycloheptane- 1 ,2-dione-bis- Nickelous chelate of cycloheptane-l,2-dione-thiosemicarbazone To a dimethylformamide solution (25 ml.) containing cycloheptane-l,2-dione-bis-thiosemicarbazone 2.7 g., 0.01 M) is added an aqueous solution of NiCl -H,O (2.4 g.. 0.01 M). The solid which precipitates is filtered and dried to give a 64 percent yield of product.

EXAMPLE XVI Cobaltous chelate of cycloheptane-l,2-dione-thiosemicarbaaone To a dimethylformamide solution (25 ml.) containing cycloheptane-l,Z-dione-bis-thiosemicarbazone (2.7 g., 0.01 M) is added an aqueous solution of CoCl,'2H,O (2.37 g., 0.0] M). The solid which precipitates is filtered and dried to give a substantial yield of product.

EXAMPLE XVll The ferrous chelates of the compounds enumerated in examples V and X are prepared following the procedure outlined in example Xl wherein stoichiometric equivalent amounts of said enumerated compounds are used in lieu of cycloheptane-l.2-dione-bis-thiosemicarbazone and substantially the same results are obtained.

EXAMPLE XVlll The cupric chelates of the compounds enumerated in examples V and X are prepared following the procedure outlined in example Xll wherein stoichiometric equivalent amounts of said enumerated compounds are used in lieu of cycloheptanel,2-dione-bis-thiosemicarbazone and substantially the same results are obtained.

EXAMPLE XIX The zinc chelates of the compounds enumerated in examples V and X are prepared following the procedure outlined in example Xlll wherein stoichiometric equivalent amounts of said enumerated compounds are used in lieu of cycloheptanel,2-dione-bis-thiosemicarbazone and substantially the same results are obtained.

EXAMPLE XX The stannous chelates of the compounds enumerated in examples V and X are prepared following the procedure outlined in example XlV wherein stoichiometric equivalent amounts of said enumerated compounds are. used in lieu of cycloheptane-l,2-dione-bis-thiosemicarbazone and substantially the same results are obtained.

EXAMPLE XXI The nickelous chelates of the compounds enumerated in examples V and X are prepared following the procedure outlined in example XV wherein stoichiometric equivalent amounts of said enumerated compounds are used in lieu of cycloheptane-l,2-dione-bis-thiosemicarbazone and substantially the same results are obtained.

EXAMPLE XXll The cobaltous chelates of the compounds enumerated in examples V and X are prepared following the procedure outlined in example XVl wherein stoichiometric equivalent amounts of said enumerated compounds are used in lieu of cycloheptane-l,2-dione-bis-thiosemicarbazone and substantially the same results are obtained.

EXAMPLE XXlIl In the screening program to determine the effectiveness of the herein disclosed compounds as coccidiostats, Eimeria renella is used as the test organism, since said organism is probably the most widely occurring species. The method of evaluation consists of treating young chicks per os with the use of a needle and syringe over a 5-day schedule. The birds receive drugs at a concentration approximately equivalent to 0.1 percent in the feed once daily for 5 consecutive days. The dose used is calculated on the basis of the average amount of food that is consumed per day bya bird 10 days of age. It is calculated that 20 mg. of drug per chick per day is equivalent to 0.1 percent in the feed. The drugs are administered in the morning and subsequently inoculated with E. lenella in the afternoon. On the 2nd, 3rd, 4th, and 5th, days, the drugs are given to complete the treatment. Test animals are held until the 8th, day after inoculation, when they are sacrificed and examined for evidence of infection.

The eiiicacy of the drug is judged by its ability to prevent mortality and by comparison of the pathologic index with that of unmedicated or infected controls. Weight gains are compared with those of infected controls, uninfected controls and positive drug controls. At necropsy, the degree of pathologic involvement is expressed as the average degree of infection (A.D.l.) based on the following scheme:

0 no lesions in the cecum l slight lesion formation in the cecum 2 moderate lesion formation in the cecum 3 severe lesion formation in the cecum 4 death due to infection Compounds, which show activity in the above said evaluation, are evaluated further by the use of the conventional drug-diet method wherein other important species of Eimeria such as E. necatrix, E. acervulina, and E. maxima are also incorporated at this stage of drug development. The drug-diet method comprises administering medicated feeds prepared by the mixing of drugs at different levels in the feed, said medicated feeds introduced at least one day prior to exposure TABLE 1 Anticoceidial activity of cycloheptane-1,2-dione-bis-thiosemicarbazone in E. tenclla infected chicks (Screening) Dose, Weight Compound mgJkg. gain A.D.I. Cycloheptane-l,2-dione-bis-thiosemicart-stone 250 68 0. 4 Infected control. 36 3. 8 Weight control 92 0.0

TABLE II Anticoccidial activity of cycloheptane-l,Z-dione-bis-thiosemicarbazone in E. tilnella, E. necatriz, and E. ucervulim infected chicks (Drug-diet met d) E. tenella Dose, Weight Compound percent gain AD .I

Cycloheptane-1,2dione-bis-thiosemi- 0. 1 24 0.0 carbazone 0, 025 42 0. 0 0. 006 70 0. 0 Infected control 40 3. 2 Weight control 74 0. 0

' a E necatru Cycloheptane-l,2-dione-bis-thiosemi- 0.025 vs 0.0 carbezone 0. 012 90 0. 1 0. 006 70 0. 1 Iniected control 4. 0 Weight control 108 0. 0

E. acervulina Cycloheptane-l,Z-dione-bis-thlosemi- 0. 025 94 0. 0 carbazone.. m. 0.0i2 76 0.0 0. 006 62 0. 0 Inieeted control 5B 2. 6 Weight control 108 0.0

EXAMPLE XXIV The drug-diet method is used for the anticoccidial evaluation of the following neutral metal chelate compounds and the ensuing results are obtained:

ANTICOCCIDIAL ACTIVITY IN E. TENELLA. INFECTED CHICKS What is claimed is:

1. A method for combatting coccidiosis in poultry which comprises orally administering thereto an anticoccidial amount of a neutral metal chelate of a compound of the formula:

wherein R is hydrogen, alkyl containing from 1 to l0 carbon atoms, chloro, hydroxy, mercapto or amino; R is hydrogen or alkyl containing from one to eight carbon atoms: n is an integer of from two to 18 and R is hydrogen or alkyl containing from one to eight carbon atoms, the chelating metal of said neutral metal chelate being Fe, Zn, CO, Cu or Ni.

2. The method of claim 1 wherein R, R and R of said compound are each hydrogen and n is 5.

3. A method as in claim 1 wherein said compound is the cupric chelate of cycloheptane-l ,Z-dionc-bis-thiosemicarbazone.

4. A method as in claim 1 wherein said compound is the ferrous chelate of cycloheptane-l ,2-dione-bis-thiosemicarbazone. 

2. The method of claim 1 wherein R, R1 and R2 of said compound are each hydrogen and n is
 5. 3. A method as in claim 1 wherein said compound is the cupric chelate of cycloheptane-1,2-dione-bis-thiosemicarbazone.
 4. A method as in claim 1 wherein said compound is the ferrous chelate of cycloheptane-1,2-dione-bis-thiosemicarbazone. 